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Article High-Throughput Genetic Testing in ALS: The Challenging Path of Variant Classification Considering the ACMG Guidelines

1,2, 1,2, , 1,2 3 Serena Lattante † , Giuseppe Marangi * † , Paolo Niccolò Doronzio , Amelia Conte , Giulia Bisogni 3, Marcella Zollino 1,2 and Mario Sabatelli 3,4

1 Section of Genomic Medicine, Department of Life Sciences and Public Health, Faculty of Medicine and Surgery, Catholic University of the Sacred Heart, 00168 Roma, Italy; [email protected] (S.L.); [email protected] (P.N.D.); [email protected] (M.Z.) 2 Complex Operational Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, A. Gemelli University Hospital Foundation IRCCS, 00168 Roma, Italy 3 Adult NEMO Clinical Center, Complex Operational Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, A. Gemelli University Hospital Foundation IRCCS, 00168 Roma, Italy; [email protected] (A.C.); [email protected] (G.B.); [email protected] (M.S.) 4 Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Catholic University of the Sacred Heart, 00168 Roma, Italy * Correspondence: [email protected]; Tel.: +39-0630154606 These authors contributed equally to this work. †  Received: 14 August 2020; Accepted: 22 September 2020; Published: 24 September 2020 

Abstract: The development of high-throughput sequencing technologies and screening of big patient cohorts with familial and sporadic amyotrophic lateral sclerosis (ALS) led to the identification of a significant number of genetic variants, which are sometimes difficult to interpret. The American College of Medical Genetics and Genomics (ACMG) provided guidelines to help molecular geneticists and pathologists to interpret variants found in laboratory testing. We assessed the application of the ACMG criteria to ALS-related variants, combining data from literature with our experience. We analyzed a cohort of 498 ALS patients using massive parallel sequencing of ALS-associated genes and identified 280 variants with a minor allele frequency < 1%. Examining all variants using the ACMG criteria, thus considering the type of variant, inheritance, familial segregation, and possible functional studies, we classified 20 variants as “pathogenic”. In conclusion, ALS’s genetic complexity, such as oligogenic inheritance, presence of genes acting as risk factors, and reduced penetrance, needs to be considered when interpreting variants. The goal of this work is to provide helpful suggestions to geneticists and clinicians dealing with ALS.

Keywords: amyotrophic lateral sclerosis; ACMG guidelines; high-throughput genetic testing; panel sequencing

1. Introduction In recent years, the fast and steady development of high-throughput technologies for genetic analysis dramatically changed the diagnostic approach in medical genetics and research strategies in related scientific fields. Consequently, these methods also impacted the study of amyotrophic lateral sclerosis (ALS), a neurodegenerative adult onset disease involving motor neurons in the cerebral cortex, brainstem, and spinal cord. Genetic factors play an important role in ALS pathogenesis, especially in familial amyotrophic lateral sclerosis cases (FALS), where a Mendelian dominant inheritance

Genes 2020, 11, 1123; doi:10.3390/genes11101123 www.mdpi.com/journal/genes Genes 2020, 11, 1123 2 of 31 pattern with high penetrance is detectable. FALS represent about 10% of all cases and the genetic cause has been identified in 70% of them. Although the remaining 90% of cases occur sporadically (sporadic amyotrophic lateral sclerosis, SALS) in the general population, a genetic cause or Mendelian etiology has been identified in 11% [1]. Thanks to the advent of next-generation sequencing (NGS) technologies and to the exte